Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells.

Abstract

The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both non-encapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.